HIV infection causes generalized global immune activation involving both the innate and adaptive arms of the immune system increasing AIDS and HIV-related non-AIDS conditions (HANA), even with successful HAART treatment. Our studies demonstrate that high levels of immune activation and senescence are seen early in HIV disease and are associated with incident AIDS, cardiovascular and liver disease especially in HCV co- infected women. The overall goal of this renewal is to continue examining mechanisms and consequences of this immune hyperactivation state and its impact on accelerated disease. We will evaluate the consequences of aberrant cytokine production that may promote fibrogenesis or tissue damage and alter T cell maturation, regulation and immune function. We find that co-infected women with liver fibrosis have significantly fewer regulatory T cells and increased percentage of senescent and terminally differentiated and effector memory T cells, indicating that immune dysregulation may accelerate inflammaging and end organ disease. In this proposal, we will comprehensively investigate the effect of long-term activation and immune dysregulation on clinical outcome in a well characterized cohort of women in WIHS. Using multiparameter flow cytometry, real- time PCR and PCR array technologies, we developed platforms to systematically evaluate both immunologic and virologic factors impacting disease. We performed complex validation experiments allowing simultaneous evaluation of soluble and cellular biomarkers, molecular gene-signatures of immune activation, exhaustion, senescence and regulation, and virologic studies from one sample. Our central hypothesis is that HIV- associated immune dysregulation and HCV infection with liver dysfunction maintains this state of immune hyperactivation, leading to immune exhaustion and senescence and more rapid progression of HANA conditions, even with effective HAART. Furthermore, liver disease will play a major role in determining immunologic, virologic and clinical outcomes especially as women age and enter menopause. Our specific aims are: Specific Aim 1) Define determinants and consequences of long-term immune activation in singly and co-infected women successfully treated with HAART and Specific Aim 2) Assess gene expression patterns involved in molecular mechanisms promoting immune activation, senescence, exhaustion, and progression to clinical outcomes. For Specific Aim 1, we will longitudinally compare levels of cytokines and soluble and cellular markers of activation and immune regulation and determine impact on immune senescence/exhaustion and disease outcome. For Specific Aim 2, we will evaluate intrinsic host gene- signature pathways of immunity using real time PCR array technology and will evaluate interferon response related to immune activation, exhaustion and senescence. These studies will identify the impact of long-term activation and dysregulation on senescence and exhaustion and the role of HCV co-infection in hopes of developing treatment strategies that may prevent continued hyperactivation and the onset disease.